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Malic enzyme recombinant bacteria and construction method and application thereof

A malic enzyme and a construction method technology, applied in the biological field, can solve the problems of limiting malic acid efficiency, low enzyme activity and the like

Active Publication Date: 2017-04-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problems in the prior art are mainly reflected in the low enzymatic activity of malic enzyme reversely catalyzing pyruvate to malic acid, which limits the efficiency of producing malic acid from pyruvate. Therefore, it is necessary to adopt enzyme transformation technology to obtain pyruvate reductive carboxylase malic enzyme

Method used

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  • Malic enzyme recombinant bacteria and construction method and application thereof
  • Malic enzyme recombinant bacteria and construction method and application thereof
  • Malic enzyme recombinant bacteria and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Screening of malic enzyme mutants

[0057] Extract total RNA from Arabidopsis thaliana, reverse transcribe to obtain cDNA, and amplify to obtain ME encoding NADP-dependent malic enzyme gene 2 .

[0058] In order to obtain potential amino acid mutation sites, firstly, on the basis of establishing the 3D structure model of the enzyme, the substrates NADPH, Mn 2+ and pyruvate bound to the enzyme active center ( figure 2 ), and screened out with pyruvate as the center Amino acid residues in the range.

[0059] By comparing the amino acid sequences of 1000 malic enzymes, the amino acid conservative and non-conservative positions in the amino acid residues were determined. On the basis of the non-conservative positions, the following single point mutation libraries were constructed: E129V, E129P, E129D, V128I, V128T, V128L, V134I, S209A, S209F, S209C, S209V, R470K, R470S, R470Y, A487G, A487S, A487Y, C490A or C490S.

[0060] The above mutated gene was connecte...

Embodiment 2

[0067] Example 2 Construction of malic enzyme mutant plasmid pTrcHisA-C490S

[0068] The specific method is as follows:

[0069] C490S mutation primer is GGTCAGGCCAACAAT AGC TATATCTTTCCGGGC, where the underlined part is the mutation point, the mutant was constructed according to the instructions of the QuikChange Lightning Site-Directed Mutagenesis Kits single point mutation kit, and pTrcHisA-ME 2 As a template, high-fidelity enzymes were used to perform PCR amplification of the whole plasmid, and then digested with DpnI for 2 hours. The PCR product was introduced into JM109 competent cells by heat shock transformation method, and the obtained mutants were sequenced, and finally the mutant plasmid pTrcHisA-C490S was obtained. cells, and extract the mutant plasmid.

[0070] Then transfer the mutant plasmid into the recombinant bacteria, and then inoculate a single colony of the recombinant bacteria into 5mL / 20mL LB liquid medium, at 37°C, 200r·min -1 Cultivate overnight under...

Embodiment 3

[0076] Example 3 Construction of malic enzyme mutant plasmid pTrcHisA-A487Y

[0077] A487Y mutation primer is TATTTGCCGGGTCAG CAG AACAATTGCTATATC, where the underlined part is the mutation point, the mutant was constructed according to the instructions of the QuikChange Lightning Site-Directed Mutagenesis Kits single point mutation kit, and pTrcHisA-ME was used in the process 2 As a template, the obtained mutant plasmid pTrcHisA-A487Y was sequenced and verified.

[0078] Then transfer the mutant plasmid into the recombinant bacteria, and then inoculate a single colony of the recombinant bacteria into 5mL / 20mL LB liquid medium, at 37°C, 200r·min -1 Cultivate overnight under the same conditions to obtain seed liquid. Inoculate the seed liquid into 30mL / 250mL LB liquid medium with a 2% inoculum amount, and inoculate at 37°C, 200r min -1 Under the condition of culture, the final concentration of 100mg L was added at the beginning of the culture -1 Ampicillin, when the OD of t...

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Abstract

The invention relates to a construction method of malic enzyme recombinant bacteria, and the method comprises the following steps: a malic enzyme mutant gene can be obtained by mutation of a NADP dependent malic enzyme gene; the malic enzyme mutant gene is connected with a carrier for recombinant expression in host bacteria to obtain the malic enzyme recombinant bacteria. The invention also discloses the malic enzyme recombinant bacteria constructed by the method and a production method of L-malic acid, the production method of the L-malic acid comprises the following steps: aerobic fermentation of the malic enzyme recombinant bacteria in a fermentation medium, induction when the malic enzyme recombinant bacteria grows until OD600 is 0.4-0.8h, and microaerobic fermentation. The malic enzyme recombinant bacteria has the advantages of high catalytic capability of pyruvate carboxylation reaction, and can obviously improve the production malic acid capacity of the malic enzyme recombinant bacteria.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a malic enzyme recombinant bacterium and its construction method and application. Background technique [0002] Malic acid, scientific name is 2-hydroxysuccinic acid, as an important C4 dicarboxylic acid, malic acid is widely used. In the field of food, malic acid is the third largest food sour agent after lactic acid and citric acid; in the field of medicine, it has the functions of anti-fatigue and protecting the liver, kidney, and heart; in the field of chemical industry, L-malic acid is a cosmetic additive. It is a color retention agent and synergist in the printing and dyeing industry, a flavoring agent, cleaning agent and deodorant for toothpaste and tobacco products, as well as a soldering aid, a waste desulfurizer and an electroplating mixture. [0003] There are five microbial pathways involved in the biosynthetic metabolic pathway of L-malic acid. Pathway I: With phosphoe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/81C12N1/21C12N1/19C12P7/46C12R1/19
CPCC12N9/0006C12N15/70C12N15/81C12N2800/101C12N2800/102C12P7/46C12Y101/0104
Inventor 刘立明高聪陈修来胡贵鹏郭亮刘佳罗秋玲
Owner JIANGNAN UNIV
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